Enzymatic syntheses of oligosaccharides (such as N-acetyllactosamine (LacNAc) and its derivatives), using glycosyltransferases (for example .beta.-1.fwdarw.4-galactosyltransferase (GaIT) EC 2.4.1.38!) and nucleoside phosphate glycosides (nucleotide-sugars) as glycosyl donors have been known for a long time (Wong, C.-H., et al., J. Am. Chem. Soc. 118, 8137-8145, 1991). Wong et al. developed LacNAc syntheses in which the nucleotide-sugar, the uridine diphosphate-glucose (UDP-Glc), is regenerated in situ. See Wong, C. -H., et al., J. Org. Chem. 47, 5416-5418 (1982) and Wong, C. -H., et al. J. Org. Chem. 57, 4343-4344, (1992). As a consequence of this, it is no longer necessary in these reactions to employ the relatively costly nucleotide-sugars in stoichiometric amounts.
The LacNAc cycle developed by Elling and Kula (DE P 42 21 595 C1; Elling, L., Grothus, M., Kula, M.-R., Glycobiol. 3, 349-355, (1993)) represents an improvement compared with cycles disclosed to date, because only three enzymes have to be employed for the synthesis of LacNAc, instead of the six enzymes used in prior art methods. See FIG. 1. Such synthesized disaccharides can be used as precursors for further reactions with different transferases, for example sialyltransferases or fucosyltransferases.
The demand for nucleotide-sugars as substrates has grown in past years due to the discovery and utilization of glycosyltransferases for the preparation of oligosaccharides. It was this development which resulted in a demand to prepare these costly nucleotide sugar substrates at minimum cost in order to more easily obtain defined glycosyl structures.
One target product of these enzymatic syntheses is, for example, the tetrasaccharide sialyl-LewisX or its derivatives (Ichikawa, Y., et al., J. Am. Chem. Soc. 114, 9283-9298 (1992)), whose importance in cell-cell recognition is the subject of intensive research. See DeFrees, S., et al., J. Am. Chem. Soc. 117, 66-79, (1995).
Sucrose synthase EC 2.4.1.13! is a glycosyltransferase which is widespread, especially in plants, and whose function as catalyst for the formation of nucleotide-sugars within plant metabolism has been reviewed in Avigad, G., Encyclopedia of Plant Physiology New Series Vol. 13A, Carbohydrates I, Intracellular Carbohydrates, Springer Verlag, Berlin, 217-347, (1982). This enzyme is suitable for synthesizing nucleotide-sugars, for example UDP-, dTDP-, ADP-, CDP- and GDP-Glc (Elling, L., Grothus, M., Kula, M.-R., Glycobiol. 3, 349-355, 1993). Purification of sucrose synthase from rice (Elling, L., Kula, M.-R., J. Biotechnol. 29, 277-286, 1993), and its use for in situ regeneration of UDP-Glc has been described by Elling et al. (DE P 42 21 595 C1; Elling, L., et al., Glycobiol. 3, 349-355, 1993). The rice enzyme is a homotetrameric protein with a molecular weight of 362 kDa. The enzyme has already been employed for the preparative synthesis dTDP-Glc starting from dTDP in an enzyme membrane reactor (EMR) (Zervosen, A., et al., Angew. Chem. 106, 592-593, (1994).
The increased availability of sucrose synthase in recent years has been made possible by purification studies such as those described by Elling (DE P 42 21 595 C1; Elling, L., et al., J. Biotechnol. 29, 277-286 (1993); Elling, L., et al., Glycobiol. 3, 349-355 (1993)). The enzyme itself has been known for several years, but insufficient amounts have been available to date to permit its protein chemical properties to be investigated.
Thus, only recently have the pH optimum, and the ions necessary for the reaction (ionic strength, presence of ions indispensable for the reaction) and the regulation of this enzyme (end product inhibition) become known (L. Elling, Glycobiology 5, 201-206, 1995). In addition while the enzyme is not commercially available, the isolation procedure for sucrose synthase is described in Elling, et al. Biotechnol 29:277-286 (1993), Elling, et al. Glycobiol. 3:349-355 (1993), and DE patent 42 21 595 C1, all of which are hereby incorporated by reference.
Preparative syntheses of nucleotide-sugars starting from a nucleoside monophosphate (NMP) have already been described (Heidlas J. E., Williams K. W., Whitesides G. M., Acc. Chem. Res., 25., 307-314, 1992). Heidlas discloses the conversion of NMP to nucleoside triphosphate (NTP) using myokinase and pyruvate kinase, which then reacted with the particular sugar 1-phosphate to give the nucleotide-sugar with catalysis by a specific pyrophosphorylase. In contrast to the nucleoside-specific pyrophosphorylases used in such processes, use of the enzyme sucrose synthase makes it possible to prepare variously activated glucose donors. Hence, it has now been found that sucrose synthase is able to accept both nucleotide diphospates (NDP) and deoxy nucleotide diphosphates (dNDP) as substrates. Thus, for example, dUDP-glucose can be prepared preparatively starting from sucrose as glycosyl donor and dUDP. Coupling kinase and sucrose synthase enzymatic reactions results in less costly production of nucleotide-sugars. Nucleoside monophosphates (NMP) can be used in such a coupled synthesis and can be obtained at distinctly lower cost than nucleoside diphosphates (NDP).
Some primary nucleotide-sugars are enzymatically converted in cells into other, so-called secondary nucleotide-sugars. Thus, for example, GDP-fucose is produced from GDP-mannose in three consecutive steps. Some recent findings in this area have been worked out by Chang et al. taking the example of porcine salivary glands (J. Biol. Chem., 263, 1693-1697, 1988). In this study, the authors describe the conversion of GDP-mannose first into GDP-4-keto-6-deoxymannose using a specific dehydratase, subsequently an epimerase and reductase activity to GDP-fucose. Direct preparation of fucose starting from mannose cannot take place in metabolism without the presence of the nucleotide group. These enzymes therefore rely on an activation, i.e. the nucleotide moiety. Based on the current state of research, it is likely that the formation of other secondary nucleotide-sugars takes place in a similar fashion. The nucleotide-sugars dUDP-and dTDP-6-deoxy-D-xylo-4-hexulose are formed via catalysis using dTDP-Glc-4,6-dehydratase EC 4.2.1.46! from dUDP-and dTDP-Glc (Zarkowsky, H., Glaser, L., J. Biol. Chem. 222, 4750-4756, 1969). dTDP-6-Deoxy-D-xylo-4-hexulose is an intermediate product in the biosynthetic pathway of the dTDP-L-rhamnose. Enzymatic synthesis and isolation of such a have been described (Marumo, K., Lindqvist, L., Verma, N., Weintraub, A., Reeves, P. R., Lindberg, A. A., Eur. J. Biochem. 204, 539-545, 1992). dUDP-Glc cannot be bought but has been synthesized in analytical amounts using the dTDP-Glc pyrophosphorylase from Pseudomonas aeruginosa (Melo, A., Glaser, L., J. Biol. Chem. 240, 398-405, 1965). It is possible by utilizing the synthetic potential of sucrose synthase to prepare dUDP-and dTDP-6-deoxy-D-xylo-4-hexulose starting from dUMP and dTDP respectively (FIG. 2) (Stein, A., Dissertation, Heinrich Heine University, Dusseldorf, 1995). However, in the process taught by Stein, et al. the separate enzymatic reactions required to produce such hexuloses are carried out in separate reaction vessels. In other words, (1) the coversion of NMP to NDP, (2) the transfer of the glucose moiety to dTDP or dUDP and (3) the dehydration of glucose all take place in separate vessels and one reaction must be complete before the next step is initiated.
Thus, the prior art does not disclose a method for the synthesis of nucleotide sugars whereby all the reagents and enzymes necessary to produce a nucleotide-sugar from a NMP, sugar and phosphate source may be incubated together. In particular, the prior art does not teach a method whereby sucrose synthase can be incubated along with enzymes which require dUDP-glucose as a substrate, such as a dehydratase.
It would therefore be useful to have a method for making nucleotide-sugars in which all reagents and enzymes could be incubated together in the same reaction vessel. In particular, it would be advantageous to have a method where sucrose synthase and a dUDP-glucose dehydratase could be used together to produce nucleotide-6-deoxy-D-xylo-4-hexulose.